The present invention relates generally to materials and methods for developing protective responses against Herpes simplex virus ("HSV") disease states. More particularly, the present invention relates to novel preparations of HSV envelope glycoprotein gD which, when employed as the active immunogen of vaccine compositions, provoke significantly better protection in a recipient against an HSV infection disease state than heretofore obtainable in the art. The invention also relates to immunoreactive polypeptides which duplicate or substantially duplicate amino acid sequences extant in HSV gD and to the use of such polypeptides in vaccination procedures.
Incorporated by reference herein for purposes of providing relatively current information concerning the background of the present invention is a publication of Wise, et al., "Herpes Simplex Virus Vaccines", J. Infectious Diseases, 136, pp. 706-711 (1977). Briefly summarized, this 1977 publication states that clinical illness caused by Herpes simplex virus, and especially the disability associated with recurrent infections, is a significant health problem that cannot be prevented at present. Alteration of the immune system by vaccination, it was thought, could potentially prevent or limit the infection upon subsequent exposure to the natural virus. Because such vaccination had proved efficacious in the control of many human diseases of viral etiology, an attempt to develop a vaccine against HSV was presented as a logical consideration. To accomplish this goal satisfactorily, it was noted that a number of attributes unique to the virus must be examined. These included the natural history, epidemiology, and severity of the disease, the various immune responses that were known to follow infection with the virus or immunization with experimental vaccines, and the possible risks associated with vaccine usage.
HSV, a large, enveloped, DNA-containing virus, was noted to cause a variety of clinical entities associated with primary infection, principally involving the skin, mucosal membranes, cornea, and nervous system. The two types of HSV--type 1 (HSV-1) and type 2 (HSV-2)--were mentioned to be distinguishable by their antigenic, biologic, and biochemical characteristics. Because HSV-1 and HSV-2 differed antigenically and because an individual could have a primary infection with either type, "type-specific" HSV vaccines were stated to be a likely requirement of any vaccine development program.
HSV was noted to have the ability to cause both "primary" and "recurrent" infections. Since the pathogenesis of primary and recurrent infections were clearly different, the rationale for development of a vaccine against these two entities were considered separately.
Natural infection with HSV was noted to bring into play many specific and nonspecific components of the immune defense system. Antibodies had been found to develop soon after primary infection, reach maximal levels within three to four weeks, and remain detectable for many years thereafter. Cellular immune responses to HSV infection were also detected in vivo by a delayed-type hypersensitivity response to the intradermal injection of viral antigens and in vitro by the many correlates of cellular immunity. The effects of the immune response induced by HSV upon subsequent infections in laboratory animals and humans were reported on. For example, mice immunized with either live or killed HSV, unlike unimmunized mice, were frequently found to be resistant to subsequent lethal challenge with HSV. In humans, it appeared that if individuals had preexisting HSV-1 antibodies, primary infection with HSV-2 tended to be milder. This observation and the data from studies of HSV disease in animals suggested that the immune response induced by HSV could have a beneficial effect on subsequent HSV infections and that, if a HSV vaccine could induce a similar immune response, it could ameliorate the clinical manifestations of primary HSV infections.
Herpes simplex viruses were then noted to characteristically persist in the host and cause recurrent infections, and the disability associated with these recurrences was described as a significant health problem. The most frequent manifestations of recurrent herpetic disease states were disclosed to involve the orofacial and genital regions and recurrent herpetic keratitis was characterized as a leading cause of blindness in the United States. Herpetic genital infections with a high incidence of subsequent recurrent episodes were noted as being recognized more frequently and being associated with significant morbidity.
The source of the virus that leads to recurrent disease was noted to be of major importance to the rationale for developing a HSV vaccine. On the basis of a variety of clinical observations, it was concluded that the virus remained dormant in nervous tissue. The isolations of HSV-1 from the trigeminal ganglia and of HSV-2 from the sacral ganglia of humans were asserted to be major steps in the further development of this concept, as were the results obtained from animal models. After extensive discussion of clinical studies of latent infections, it was generally concluded that the possibility of developing a vaccine protective against both primary infection and recurrent infection was highly remote.
HSV vaccine candidates were enumerated: live attenuated virus; inactivated whole virus; and inactivated "sub-unit" viral components. Live viral vaccines were noted to be frequently preferred over inactivated ones because the immune responses induced by live vaccines tend to be higher and of longer duration, and because live vaccines require a smaller inoculum owing to the ability of the virus to multiply in the host. The disadvantage of live viral vaccines in terms of difficulty in production and in maintenance in proper degree of attenuation were noted as were the then-preliminary studies revealing that at least HSV-2 appeared to be oncogenic in humans. Since it appeared that infectious virus was not required for the in vitro transformation of cells, this highly unfavorable risk consideration was also held to be applicable to inactivated vaccines containing viral nucleic acid. Various live and attenuated virus vaccine preparations were discussed and the conclusion was reached that none provided beneficial results sufficient to justify oncogenic risks.
The development of an inactivated vaccine containing sub-unit viral components with little or no viral DNA was therefore proposed as lessening the concern of oncogenicity. Sub-unit component vaccines, however, were noted to require difficult purification processes and to have the disadvantage of usually being poor immunogens. Concern was also expressed that subsequent vaccine-induced immunity may not only fail to protect against natural virus challenge but, as in the case of inactivated measles vaccine, could conceivably cause a more severe clinical illness upon exposure to the natural virus.
The 1977 publication concluded that, while vaccination was one possible method for attaining the goal of prophylaxis, as of that date the efforts aimed at development of a HSV vaccine that was clinically acceptable and of proven efficacy were completely unsuccessful.
Since the time of the above-noted publication, the oncogenicity of Herpes simplex virus DNA and RNA has been the subject of confirmation by a number of investigators. See, e.g., Rapp, "Transformation by the Herpes Simplex Viruses", pp. 221-227 in "The Human Herpesviruses, An Interdisciplinary Perspective", Nahmias, et al., eds., Elsevier North Holland, Inc., New York, N.Y. (1981) and the publications cited therein. Such studies have essentially eliminated any remaining prospect for widespread use of live virus vaccines as well as those vaccine compositions including assertedly nonpathogenic, attenuated HSV strains as illustrated in U.S. Pat. No. 3,897,549.
Consistent with the general recognition of the desirability of vaccine compositions which exclude Herpes simplex virus DNA and RNA, the number of proposals for so-called "sub-unit" vaccines has increased. See, generally, Moreschi, et al., "Prevention of Herpes Simplex Virus Infections", pp. 440-445 in "The Human Herpesvirus, An Interdisciplinary Perspective", Nahmias, et al., eds., Elsevier North Holland, Inc., New York, N.Y. (1981). As one example, U.S. Pat. No. 4,158,054 proposes, but does not exemplify, a Herpes simplex sub-unit vaccine prepared by introducing inactivated whole virus particles into continuous loading zonal ultracentrifugation provided with a density gradient containing a haemolytic surfactant followed by binding of "split" sub-units isopycnically. As other examples, there may be noted the nucleic acid freed vaccines described by: Cappel, Archives of Virology, 52, pp. 29-35 (1976); Kitces, et al., Infection and Immunity, 16, pp. 955-960 (1977); Slichtova, et al., Archives of Virology, 66, pp. 207-214 (1980); and Skinner, et al., Med. Microbiol. Immunol., 169, pp. 39-51 (1980). All the vaccine compositions of the foregoing publications were prepared by separative methodologies wherein greater or lesser care was taken to limit or eliminate nucleic acids from the fractions extracted. None of the vaccines, however, has been found to provide uniform protection of all vaccinate test animals from death by lethal challenge with Herpes simplex virus, a generally recognized requisite for continued evaluation.
Another Herpes simplex vaccine recently proposed and relatively thorougly tested is a composition prepared by using what is asserted to be a viral glycoprotein sub-unit fraction. In Hilleman, et al., "Sub-unit Herpes Simplex Virus-2 Vaccine" pp. 503-506 in "The Human Herpesviruses, An Interdisciplinary Perspective" Nahmias, et al., eds., Elsevier North Holland, Inc., New York, N.Y. (1981), there is proposed a mixed glycoprotein sub-unit vaccine prepared using chick embryo fibroblasts infected with type 2 Herpes simplex virus. Briefly put, the vaccine antigen is prepared through glycoprotein release by treatment of infected cells with Triton X-100, digestion with DNase, purification on a lectin affinity column, and chromatography on Sephadex. The material is then treated with formalin and formulated in alum adjuvant. Vaccinated mice are noted to be protected against lethal challenge with Herpes simplex virus type 2 to a significantly greater degree than the alum adjuvant-treated controls. The glycoprotein was less effective in reducing mortality, however, than an aqueous, UV-inactivated whole virus vaccine (which itself did not prevent death in all vaccinated animals). The ability of the glycoprotein vaccine to induce formation of both homologous and heterologous type antibodies in humans was acknowledged to be limited, and cell mediated immunity assays with respect to homologous and heterologous types indicated both limited and transitory effects.
Of significant interest to the background of the present invention is the extensive body of information developed over the years concerning the major envelope glycoproteins of HSV. An extensive and extremely well-annotated monograph on this topic is presented in Norrild, "Immunochemistry of Herpes Simplex Virus Glycoproteins," in Current Topics in Microbiology and Immunology, 90: pp. 67-106, Springer Verlag, Berlin (1980). The major topics of discussion are: the structue, synthesis and function of HSV-specified glycoproteins; the immunological reactivity of viral membrane proteins and their components; and demonstrations of the antigenic specificities of antibodies to individual glycoproteins.
Briefly summarized, the publication notes that Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) specify at least five major glycoproteins, designated gA, gB, gC, gD and gE, which are to be found not only in the envelope of virus particles, but in the plasma membrane of infected cells and in detergent-treated cytoplasmic extracts derived from infected cells. These glycoproteins carry strong antigen determinants that include production of antibodies in an infected host organism, and they appear to be the major immunochemical stimuli at both humoral and cellular levels in the host. Some of the viral antigen determinants are in common (i.e., gB and gD), while some are specific for one or the other of the two virus types (i.e., gC and gE). [See also, Spear, "Herpes Viruses," pp. 709-750 in "Cell Membranes and Viral Envelopes, Vol. 2," Blough, et al., eds., Academic Press, New York, N.Y. (1980)]
Of even greater significance to the background of the present invention are the publications of one or both of the co-inventors and their co-workers which have, commencing in 1972, provided a most substantial portion of all available information concerning one of the HSV envelope glycoproteins, gD. Incorporated herein by reference, therefore, are the following:
(1) Cohen, et al., J. Virol., 10: pp. 1021-1030 (1972); PA0 (2) Ponce de Leon, et al., J. Virol., 12: pp. 766-774 (1973); PA0 (3) Cohen, et al., J. Virol., 14: pp. 20-25 (1974); PA0 (4) Cohen, et al., J. Virol., 27: pp. 172-181 (1978); PA0 (5) Eisenberg, et al., J. Virol., 31: pp. 608-620 (1979); PA0 (6) Eisenberg, et al., J. Virol., 35: pp. 428-435 (1980); and PA0 (7) Cohen, et al., J. Virol., 36: pp. 429-439 (1980).
The studies reported in the above-noted publications of the co-inventors and their co-workers have focused on gD of HSV-1 ("gD-1") and, in particular, on the isolation, purification and characterization of this glycoprotein. Using an extensive series of chromatographic steps, native gD-1 (previously known as CP-1 antigen) was purified in quantities sufficient to develop a monoprecipitin (or polyclonal) anti-CP-1 serum which had high titers of type-common neutralizing activity. Using anti-CP-1 as an immunological probe, it was demonstrated that gD-1 and the gD of HSV-2 ("gD-2") are both processed from lower molecular weight precursors to higher molecular weight product forms in infected cells by addition of oligosaccharides. Significant structural similarities between gD-1 and gD-2 were established by tryptic peptide analysis. Moreover, gD-1 was shown to be structurally identical whether isolated from infected human (KB) or from hamster (BHK21) cells.
Of considerable interest were the above-noted reports of the ability of the chromatographically purified gD-1 to provoke, in vivo, the generation of serum neutralizing antibodies which were fully protective of cells in culture against both HSV-1 and HSV-2 infections, as well as the ability of gD-1 to "block" HSV-1 and HSV-2 virus infection neutralization by protective sera.
Finally, recent studies have described the preparation and properties of several monoclonal antibodies to HSV glycoprotein gD and other HSV glycoproteins. One report of such a study [Dix, et al., Infection and Immunity, 34: pp. 192-199 (1981)] notes that certain monoclonal antibodies to gD-1 and gC-1 were capable of use in conferring passive immunological protection against lethal challenge with HSV-1. Passive immunization with a monoclonal antibody to gD-1 (termed "HD-1") was also attributed with providing protection with a lethal challenge with HSV-2.
Along with the above-described need for vaccine preparations for use in prevention and treatment of Herpes simplex virus disease states, there additionally exists a need for rapid and specific diagnostic tests for Herpes virus diseases and, more specifically, for antigenic substances useful in fluorescence, immunoperoxidase labelling, radioimmune and enzyme-linked immunoabsorbant assays. Such assays are commonly employed, for example, in the detection of Herpes simplex virus antibodies in samples of body fluids such as spinal fluids taken from those patients suspected of having encephalitis of Herpes simplex virus origin. See, e.g., Sever, "The Need for Rapid and Specific Tests for Herpesviruses," pp. 379-380 in "The Human Herpesviruses, An Interdisciplinary Perspective," Nahmias, et al., eds., Elsevier North Holland, Inc., New York, N.Y. (1981).
Subsequent to the Feb. 18, 1982 filing of applicants' copending U.S. patent application Ser. No. 350,021, Watson, et al. carried out nucleic acid sequencing studies of a protein coding region of the HSV-1 (Patton strain) genome corresponding to gD-1. The results of this work appear in Science, 218, pp. 381-384 (1982). Based on the nucleic acid sequence ascertained in these studies, Watson, et al. provided a putative 394-amino acid sequence for gD-1, indicating likely glycosylation sites, designating the first twenty amino acids at the amino terminal as a putative "signal" peptide, and noting the likelihood that a series of 25 amino acids at the carboxy terminal was involved in anchoring the glycoprotein to other membrane components. DNA vectors, neither of which included the first fifty-two codons (156 bases) of the published DNA sequence, were constructed for use in microbial expression of a "gD-related" polypeptide and a .beta.-galactosidase/gD-1-related fusion polypeptide. Watson, et al. further reported that rabbits injected with the fusion protein product of E. coli expression of the fusion gene produced neutralizing antibodies to both HSV-1 and HSV-2. The directly-expressed polypeptide was not tested in vivo but was screened by immunoprecipitation assay against certain of the seventeen monoclonal antibodies screened for neutralization and RIP activity by the applicants and their co-workers in Eisenberg, et al., J. Virol., 41, pp. 478-488 (1982). The directly-expressed gD-related polypeptide was noted to be immunoprecipitable by monoclonal antibodies of Groups, I, IV and V (type common 4S, type 1 specific 1S, and RIP type 1 specific 55S and 57S) as well as polyclonal anti-HSV-1 rabbit antiserum. The polypeptide was reportedly not immunoprecipitated by monoclonals of Groups II and III (RIP type-common 12S and type-common 11S) or the group-undesignated monoclonal antibody 50S.